Removal of intervening sequences (splicing) is a requirement common to the expression of all classes of genes. Although similarities among other classes of splicing reactions are consistent with common origin, the unique features of the tRNA splicing reaction suggest a separate origin or early divergence. Thus an analysis of the tRNA splicing reaction will shed light not only on the role of splicing in the expression of this essential class of genes but also on the potential origins of tRNA gene introns, their significance in the evolution of tRNA gene families and the relationship of tRNA splicing to other classes of splicing reactions. Splicing of tRNA precursors in extracts of Saccharomyces cerevisiae requires two activities; a membrane-bound endonuclease and a soluble, ATP-independent RNA ligase. The proposed experiments will examine the interaction of the splicing enzymes with their substrates. These experiments can be divided into two categories. The first is an anaysis of elements of pre-tRNA sequence and structure required for splicing. Experiments in this category include construction of tRNA gene mutations and inhibition of splicing by chemical modification of tRNA substrates. The goal in this area is a precise definition of the role that each element of the substrate plays in splicing. A complete description of the tRNA splicing reaction will also require analysis of the properties of the splicing enzymes. In the past, the inability to obtain large quantities of purified endonuclease has greatly hindered progress in this field. The second experimental category is an analysis of yeast mutants defective in endonuclease activity. The immediate goals are to define the basis for the splicing defects in this mutants. The long term goal is to provide us with the means for analyzing the structure, properties and substrate interactions of the tRNA endonuclease.